Fc fusion proteins of human granulocyte colony-stimulating factor with increased biological activities

ABSTRACT

Fc fusion proteins of human G-CSF with increased biological activities relative to rhG-CSF on a molar basis are disclosed. The hG-CSF-L-vFc fusion protein comprises hG-CSF, a flexible peptide linker of about 20 or fewer amino acids, and a human IgG Fc variant. The Fc variant is of a non-lytic nature and shows minimal undesirable Fc-mediated side effects. A method is also disclosed to make or produce such fusion proteins at high expression levels. Such hG-CSF-L-vFc fusion proteins exhibit extended serum half-life and increased biological activities, leading to improved pharmacokinetics and pharmacodynamics, thus fewer injections will be needed within a period of time.

BACKGROUND

Granulocyte colony-stimulating factor (G-CSF) is a 20 kilodalton (kDa)glycoprotein that promotes the proliferation of progenitor cells andinduces their differentiation into neutrophils. In addition, G-CSFprolongs the survival of mature neutrophils and activates theirfunctions Human G-CSF (hG-CSF) is produced by monocytes, macrophages,fibroblasts and endothelial cells (see, for example, Moore, Annu. Rev.Immunol., 9:159–191, 1991; Nicola, Annu. Rev. Biochem., 58:45–77, 1991).The biological effects of G-CSF are mediated through its interactionwith the G-CSF receptor (G-CSF-Rc) expressed on the surface of bonemarrow hematopoietic progenitors and cells of the myeloid lineage. Uponbinding G-CSF, the receptor is activated and undergoes homodimerization,followed by phosphorylation of Janus family of tyrosine kinases.Subsequently, a series of intracellular signal transduction events takeplace, leading to the increase of the number of progenitor cells, theirmaturation into neutrophils, and further activation of effectorfunctions in mature neutrophils (see, for example, Demetri et al.,Blood, 78:2791–2808, 1991). Therefore, G-CSF plays an essential role notonly in the regulation and maintenance of hematopoiesis, but also inhost defense against infection and inflammation.

Recombinant human G-CSF (rhG-CSF) is widely used in the treatment ofpatients with neutropenia as a result of receiving chemotherapy.Administration of rhG-CSF is effective in restoring functioningneutrophils to these patients, leading to a decrease ofinfection-related events. Use of rhG-CSF allows intensified dosing orscheduling of chemotherapeutic agents that may be of benefit to cancerpatients. Besides chemotherapy-induced neutropenia, rhG-CSF has beenused for the treatment of myelosuppression after bone marrowtransplantation, acute leukemia, aplastic anemia, myelodysplasticsyndrome, severe chronic neutropenias, and mobilization of peripheralblood progenitor cells for transplantation (see, for example, Welte etal., Blood, 88:1907–1929, 1996).

The elimination half-life of the serum concentration of rhG-CSF isapproximately 3 to 4 h for intravenous or subcutaneous administration.The safety profile and patient tolerance of rhG-CSF are good withmedullary bone pain being the only frequent and significant side effect.The relatively low toxicity of rhG-CSF has made it feasible to developlonger-acting derivatives to decrease the inconvenience of the daily ortwice-daily injection schedule. Attachment of polyethylene glycol (PEG)to various proteins, including G-CSF, has been reported to yieldderivatives with higher in vivo potency due to their longer half-lives(see, for example, Zalipsky et al., in “PEG chemistry: biotechnical andbiomedical applications”, pp. 347–370, 1992). PEG-conjugated proteinsusually have considerably lower in vitro biological activity than theirunmodified parent proteins (Eliason et al., Stem Cells, 18:40–45, 2000).The increased in vivo potency of these modified proteins is, at least inpart, due to decreased removal by the kidney in a manner proportional totheir molecular weight (Yamaoda et al., J. Pharmaceut. Sci., 83:601–606,1994). We unexpectedly discover that it is possible to increase thepotency of hG-CSF through prolonging its half-life as well as enhancingits biological activity is to attach the Fc region derived from humanIgG at the C-terminus of hG-CSF, as described in this invention.

Immunoglobulins of IgG class are among the most abundant proteins inhuman blood. Their circulation half-lives can reach as long as 21 days.Fusion proteins have been reported to combine the Fc regions of IgG withthe domains of another protein, such as various cytokines and solublereceptors (see, for example, Capon et al., Nature, 337:525–531, 1989;Chamow et al., Trends Biotechnol., 14:52–60, 1996); U.S. Pat. Nos.5,116,964 and 5,541,087). The prototype fusion protein is a homodimericprotein linked through cysteine residues in the hinge region of IgG Fc,resulting in a molecule similar to an IgG molecule without the CHIdomains and light chains. Due to the structural homology, Fc fusionproteins exhibit in vivo pharmacokinetic profile comparable to that ofhuman IgG with a similar isotype. This approach has been applied toseveral therapeutically important cytokines, such as IL-2 andIFN-α_(2a), and soluble receptors, such as TNF-Rc and IL-5-Rc (see, forexample, U.S. Pat. Nos. 5,349,053 and 6,224,867). It is desirable toextend the circulating half-life of G-CSF and/or to increase itsbiological activity by making fusion proteins containing G-CSF linked tothe Fc portion of the human IgG protein as disclosed and/or described inthis invention.

Erythropoietin (EPO) derivatives, such as dimers, have been reported.Relative to the EPO monomer, a fusion protein consisting of two completeEPO domains separated by a 3- to 7-amino acid peptide linker exhibitedreduced activity (Qiu et al., J. Biol. Chem., 273:11173–11176, 1998).However, when the peptide linker between the two EPO domains was 17amino acids in length, the dimeric EPO molecule exhibited considerablyenhanced in vitro and in vivo activities (see, for example, Sytkowski etal., J. Biol. Chem., 274:24773–24778, 1999; U.S. Pat. No. 6,187,564).The length of the peptide linker between the two hematopoietic growthfactors is important, while not bound by this theory, presumably due toits effect on the flexibility of such molecular forms. We find that thisapproach is generally applicable to other therapeutic proteins,including G-CSF. We'll also refer this to this as a flexible peptidelinker.

In most of the reported Fc fusion protein molecules, a hinge regionserves as a spacer between the Fc region and the cytokine or solublereceptor at the amino-terminus, allowing these two parts of the moleculeto function separately (see, for example, Ashkenazi et al., CurrentOpinion in Immunology, 9:195–200, 1997). A human G-CSF fusion proteinwith an appropriate peptide linker between the hG-CSF and Fc moieties(hG-CSF-L-Fc) is more active than rhG-CSF, with in vitro activity atleast 2-fold as that of rhG-CSF on a molar basis. It is discoveredaccording to this invention that an added peptide linker present betweenhG-CSF and a human IgG Fc variant enhances the in vitro biologicalactivity of the hG-CSF-L-Fc molecule in two ways: (1) keeping the Fcregion away from the G-CSF-Rc binding sites on G-CSF, and (2) keepingone G-CSF from the other G-CSF domain, so both G-CSF domains caninteract with G-CSF-Rc on the granulocyte procursor cells independently.For the present invention, a flexible peptide linker of about 20 orfewer amino acids in length is preferred. More preferably, the peptidelinker should have at least two amino acids in length. Furthermore, itis even more preferable to use a peptide linker comprising two or moreof the following amino acids: glycine, serine, alanine, and threonine.

The Fc region of human immunoglobulins plays a significant role inimmune defense for the elimination of pathogens. Effector functions ofIgG are mediated by the Fc region through two major mechanisms: (1)binding to the cell surface Fc receptors (Fc_(γ)Rs) can lead toingestion of pathogens by phagocytosis or lysis by killer cells via theantibody-dependent cellular cytotoxicity (ADCC) pathway, or (2) bindingto the C1q part of the first complement component C1 initiates thecomplement-dependent cytotoxicity (CDC) pathway, resulting in the lysisof pathogens. Among the four human IgG isotypes, IgG1 and IgG3 areeffective in binding to Fc_(γ)R. The binding affinity of IgG4 to Fc_(γ)Ris an order of magnitude lower than that of IgG1 or IgG3, while bindingof IgG2 to Fc_(γ)R is below detection. Human IgG1 and IgG3 are alsoeffective in binding to C1q and activating the complement cascade. HumanIgG2 fixes complement poorly, and IgG4 appears quite deficient in theability to activate the complement cascade (see, for example, Jefferiset al., Immunol. Rev., 163:59–76, 1998). For therapeutic use in humans,it is essential that when hG-CSF-L-Fc binds to G-CSF-Rc on the surfaceof the progenitor cells or other cells of the myeloid lineage, the Fcregion of the fusion protein will not mediate undesirable effectorfunctions, leading to the lysis or removal of these cells. Accordingly,the Fc region of hG-CSF-L-Fc must be of a non-lytic nature, i.e., the Fcregion must be inert in terms of binding to Fc_(γ)Rs and C1q for thetriggering of effector functions. It is clear that none of the naturallyoccurring IgG isotypes is suitable for use to produce the hG-CSF-L-Fcfusion protein. To obtain a non-lytic Fc, certain amino acids of thenatural Fc region have to be mutated for the attenuation of the effectorfunctions.

By comparing amino acid sequences of human and murine IgG isotypes, aportion of Fc near the N-terminal end of the CH2 domain is implicated toplay a role in the binding of IgG Fc to Fc_(γ)Rs. The importance of amotif at positions 234 to 237 has been demonstrated using geneticallyengineered antibodies (see, for example, Duncan et al., Nature,332:563–564, 1988). The numbering of the amino acid residues isaccording to the EU index as described in Kabat et al. (in Sequences ofProteins of Immunological Interest, 5^(th) Edition, United StatesDepartment of Health and Human Services, 1991). Among the four human IgGisotypes, IgG1 and IgG3 bind Fc_(γ)Rs the best and share the sequenceLeu234-Leu-Gly-Gly237 (only IgG1 is shown in FIG. 1). In IgG4, whichbinds Fc_(γ)Rs with a lower affinity, this sequence contains a singleamino acid substitution, Phe for Leu at position 234. In IgG2, whichdoes not bind Fc_(γ)Rs, there are two substitutions and a deletionleading to Val234-Ala-Gly237 (FIG. 1). To minimize the binding of Fc toFc_(γ)R and hence the ADCC activity, Leu235 in IgG4 has been replaced byAla (see, for example, Hutchins et al., Proc. Natl. Acad. Sci. USA,92:11980–11984, 1995). IgG1 has been altered in this motif by replacingGlu233-Leu-Leu235 with Pro233-Val-Ala235, which is the sequence fromIgG2. This substitution resulted in an IgG1 variant devoid ofFc_(γ)R-mediated ability to deplete target cells in mice (see, forexample, Isaacs et al., J. Immunol., 161: 3862–3869, 1998).

A second portion that appears to be important for both Fc_(γ)R and C1qbinding is located near the carboxyl-terminal end of CH2 domain of humanIgG (see, for example, Duncan et al., Nature, 332:738–740, 1988). Amongthe four human IgG isotypes, there is only one site within this portionthat shows substitutions: Ser330 and Ser331 in IgG4 replacing Ala330 andPro331 present in IgG1, IgG2, and IgG3 (FIG. 1). The presence of Ser330does not affect the binding to Fc_(γ)R or C1q. The replacement of Pro331in IgG1 by Ser virtually abolished IgG1 ability to C1q binding, whilethe replacement of Ser331 by Pro partially restored the complementfixation activity of IgG4 (see, for example, Tao et al., J. Exp. Med.,178:661–667, 1993; Xu et al., J. Biol. Chem., 269:3469–3474, 1994).

We discover that at least three Fc variants (vFc) can be designed and/orused for the production of hG-CSF-L-vFc fusion proteins (FIG. 1). HumanIgG2 Fc does not bind Fc_(γ)R but showed weak complement activity. AnFc_(γ2) variant with Pro331 Ser mutation should have less complementactivity than natural Fc_(γ2) while remain as a non-binder to Fc_(γ)R.IgG4 Fc is deficient in activating the complement cascade, and itsbinding affinity to Fc_(γ)R is about an order of magnitude lower thanthat of the most active isotype, IgG1. An Fc_(γ4) variant with Leu235Alamutation should exhibit minimal effector functions as compared to thenatural Fc_(γ4). The Fc_(γ1) variant with Leu234Val, Leu235Ala andPro331 Ser mutations also will exhibit much less effector functions thanthe natural Fc_(γ1). These Fc variants are more suitable for thepreparation of the G-CSF fusion proteins than naturally occurring humanIgG Fc. It is possible that other replacements can be introduced for thepreparation of a non-lytic Fc without compromising the circulatinghalf-life or causing any undesirable conformational changes.

There are many advantages with the present invention. The increasedactivity and prolonged presence of the hG-CSF-L-vFc fusion protein inthe serum can lead to lower dosages as well as less frequent injections.Less fluctuations of the drug in serum concentrations also meansimproved safety and tolerability. Less frequent injections may result inbetter patient compliance and quality of life. The hG-CSF-L-vFc fusionprotein containing a non-lytic Fc variant will therefore contributesignificantly to the management of a variety of conditions associatedwith an impaired immune or hematopoietic system, including cancerchemotherapy, leukemias, anemias AIDS, bone marrow transplantation, andchronic neutropenias.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to an hG-CSF-L-vFc fusionprotein. This hG-CSF-L-vFc fusion protein comprises hG-CSF, a peptidelinker (denoted by L), and a human IgG Fc variant (denoted by vFc). Itis preferable to use a flexible peptide linker of about 20 or fewer,more preferably to about 2, amino acids in length and the flexiblepeptide linker contains or comprises of two or more of amino acidsselected from the group consisting of glycine, serine, alanine, andthreonine. The IgG Fc variant is of non-lytic nature and contains aminoacid mutations as compared to naturally occurring IgG Fc.

It is another embodiment of the present invention that the human Ig Fccomprises a hinge, CH2, and CH3 domains of human IgG, such as humanIgG1, IgG2, and IgG4. The CH2 domain contains amino acid mutations atpositions 228, 234, 235, and 331 (defined by the EU numbering system).It is believed that these amino acid mutations serve to attenuate theeffector functions of Fc.

In yet another embodiment of the present invention, a method isdisclosed for making or producing such recombinant fusion proteins froma mammalian cell line such as a CHO-derived cell line. Growingtransfected cell lines under conditions such that the recombinant fusionprotein is expressed in its growth medium in excess of 10, preferably30, μg per million cells in a 24 hour period. These hG-CSF-L-vFc fusionproteins are characterized by and exhibit increased/enhanced biologicalactivity, preferably at least two fold (2×) in vitro activity, on amolar basis, relative to that of rhG-CSF and extended serum half-lifewithout undesirable side effects, leading to improved pharmacokineticsand pharmacodynamics, thus lower dosages and fewer injections would beneeded to achieve similar efficacies.

A further embodiment of the present invention provides a method formaking a recombinant fusion protein comprising hG-CSF, a flexiblepeptide linker, and a human IgG Fc variant, which method comprises: (a)generating a CHO-derived cell line; (b) growing the cell line underconditions the recombinant fusion protein is expressed in its growthmedium in excess of 10 μg, preferably 30 μg, per million (10⁶) cells ina 24 hour period; and (c) purifying the expressed protein from step (b),wherein the recombinant fusion protein is characterized by and exhibitsan enhanced in vitro biological activity of at least 2 fold (2×)relative to that of rhG-CSF on a molar basis. In this case, preferably,the flexible peptide linker containing or comprising about 20 or fewer,but not fewer than 2, amino acids is present between hG-CSF and thehuman IgG Fc variant; and the flexible peptide linker comprises two ormore amino acids selected from the group consisting of glycine, serine,alanine, and threonine; and wherein the human IgG Fc variant comprises ahinge, CH2, and CH3 domains selected from the group consisting of humanIgG2 with Pro331Ser mutation, human IgG4 with Ser228Pro and Leu235Alamutations, and human IgG1 with Leu234Val, Leu235Ala, and Pro331Sermutations.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 shows the amino acid sequence alignment from the hinge and CH2regions of human IgG1, IgG2, IgG4 and their variants. Three portions arecompared: amino acid position 228, 234–237, and 330–331. Amino acidmutations of the variants are indicated in bold italics. The EUnumbering system is used for the amino acid residues.

FIG. 2 shows the nucleotide sequence and deduced amino acid sequence of(A) hG-CSF-L-vFc_(γ2), (B) hG-CSF-L-vFc_(γ4), and (C) hG-CSF-L-vFc_(γ1),as the HindIII-EcoRI fragment in the respective phGFP expression vector.Amino acid residues −30 to −1 is the leader peptide of human G-CSF. Themature protein contains human G-CSF (amino acid residues 1 to 174), apeptide linker (amino acid residues 175 to 190), and a Fc variant (aminoacid residues 191 to 418 of vFc_(γ2), 191 to 419 of vFc_(γ4), and 191 to417 of vFc_(γ1)). In the Fc regions, nucleotide and corresponding aminoacid mutations in bold are also underlined.

DETAILED DESCRIPTION OF THE INVENTION

1. Construction of the Gene Encoding the hG-CSF-L-vFc_(γ2) FusionProtein

A fusion protein is assembled from several DNA segments. The geneencoding the leader peptide and mature protein of human G-CSF isobtained by reverse transcription and polymerase chain reaction (PCR)using RNA prepared from the human bladder carcinoma 5637 cell line. Forthe convenience of cloning, SEQ ID NO: 1 (Table 1), which incorporates arestriction enzyme cleavage site (HindIII) is used as the 5′oligonucleotide primer. Table 1 shows the sequences of oligonucleotidesused for the cloning of the hG-CSF-L-vFc fusion proteins. The 3′ primer(SEQ ID NO:2) eliminates the G-CSF termination codon and incorporates aBamHI site. The resulting DNA fragments of approximately 600 bp inlength are inserted into a holding vector such as pUC19 at the HindIIIand BamHI sites to give the phGCSF plasmid. The sequence of the humanG-CSF gene is confirmed by DNA sequencing.

The gene encoding the Fc region of human IgG2 (Fc_(γ2)) is obtained byreverse transcription and PCR using RNA prepared from human leukocytesand appropriate 5′ (SEQ ID NO:3) and 3′ (SEQ ID NO:4) primers. ResultingDNA fragments of Fcy_(γ2) containing complete sequences of the hinge,CH2, and CH3 domains of IgG2 will be used as the template to generatethe FCy_(γ2) Pro331Ser variant (vFc_(γ2)) in which Pro at position 331of Fc_(γ2) is replaced with Ser. To incorporate this mutation, twosegments are produced and then assembled by using the natural Fcy_(γ2)as the template in overlapping PCR. The 5′ segment is generated by usingSEQ ID NO:3 as the 5′ primer and SEQ ID NO:5 as the 3′ primer. The 3′segment is generated by using SEQ ID NO:6 as the 5′ primer and SEQ IDNO:4 as the 3′ primer. These two segments are then joined at the regioncovering the Pro331Ser mutation by using SEQ ID NO:7 as the 5′ primerand SEQ ID NO:4 as the 3′ primer. The SEQ ID NO:7 primer containssequences encoding a 16-amino acid Gly-Ser peptide linker including aBamHI restriction enzyme site. The resulting DNA fragments ofapproximately 700 bp in length are inserted into a holding vector suchas pUC19 at the BamHI and EcoRI sites to give the pL-vFc_(γ2) plasmid.The sequence of the gene is confirmed by DNA sequencing.

To prepare the hG-CSF-L-vFc_(γ2) fusion gene, the hG-CSF fragment isexcised from the phGCSF plasmid with HindIII and BamHI and is purifiedby agarose gel electrophoresis. The purified fragment is then insertedto the 5′-end of the peptide linker in the pL-vFcγ2 plasmid to give thephG-CSF-L-vFcγ2 plasmid. The fusion gene comprises hG-CSF, a Gly-Serpeptide linker and the Fc_(γ2) variant gene.

The presence of a peptide linker, preferably a flexible linker, between(and chemically bound to both) the hG-CSF and Fc moieties increases theflexibility of the hG-CSF domains and enhances its biological activity.For the present invention, a peptide linker of about 20 or fewer aminoacids in length is preferred. While a single amino acid is within thescope of the present invention, it is preferred to have a flexiblepeptide linker of about 20 to about 2 amino acids in length. Peptidelinker containing or comprising of two or more of amino acids selectedfrom the group consisting of glycine, serine, alanine, and threonine canbe used preferably. An example of the peptide linker contains Gly-Serpeptide building blocks, such as GlyGlyGlyGlySer. FIG. 2A shows a fusiongene containing sequences encoding hG-CSF, a 16-amino acid peptidelinker (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer, SEQ ID NO:23), and the Fcy_(γ2) Pro331Ser variant, and its corresponding aminoacid sequence (SEQ ID NO: 18).

The complete gene encoding the hG-CSF-L-vFc fusion protein is theninserted at the HindIII and EcoRI sites of a mammalian expressionvector, such as pcDNA3 (Invitrogen). The final expression vectorplasmid, named phGFP2, contains the cytomegalovirus early genepromoter-enhancer that is required for high level expression inmammalian cells. The plasmid also contains selectable markers to conferampicillin resistance in bacteria, and G418 resistance in mammaliancells. In addition, the phGFP2 expression vector contains thedihydrofolate reductase (DHFR) gene to enable the co-amplification ofthe hG-CSF-L-vFcγ2 fusion gene and the DHFR gene in the presence ofmethotrexate (MTX) when the host cells are deficient in the DHFR geneexpression (see, for example, U.S. Pat. No. 4,399,216).

2. Construction of the Gene Encoding the hG-CSF-L-vFc_(γ4) FusionProtein

Human IgG4 is observed partly as half antibody molecules due to thedissociation of the inter-heavy chain disulfide bonds in the hingedomain. This is not seen in the other three human IgG isotypes. A singleamino acid substitution replacing Ser228 with Pro, which is the residuefound at this position in IgG1 and IgG2, leads to the formation of IgG4complete antibody molecules (see, for example, Angal et al., Molec.Immunol., 30:105–108, 1993; Owens et al., Immunotechnology, 3:107–116,1997; U.S. Pat. No. 6,204,007). The Fc_(γ4) variant containing Leu235Alamutation for the minimization of FcR binding will also give rise to ahomogeneous fusion protein preparation with this additional Ser228Promutation.

The gene encoding the Fc region of human IgG4 (Fc_(γ4)) is obtained byreverse transcription and PCR using RNA prepared from human leukocytesand appropriate 5′ primer (SEQ ID NO:8) and 3′ primer (SEQ ID NO:9).Resulting DNA fragments of Fcy_(γ4) containing complete sequences of thehinge, CH2, and CH3 domains of IgG4 is used as the template to generatethe Fcy_(γ4) variant with Ser228Pro and Leu235Ala mutations (vFc_(γ4))in which Ser228 and Leu235 have been replaced with Pro and Ala,respectively. The CH2 and CH3 domains are amplified using the 3′ primer(SEQ ID NO:9) and a 5′ primer containing the Leu235Ala mutation (SEQ IDNO:10). This amplified fragment, together with a syntheticoligonucleotide of 60 bases in length (SEQ ID NO:11) containing bothSer228Pro and Leu235Ala mutations, are joined in PCR by using SEQ IDNO:12 as the 5′ primer and SEQ ID NO:9 as the 3′ primer. The SEQ IDNO:12 primer contains sequences encoding a 16-amino acid Gly-Ser peptidelinker including the BamHI site. The resulting DNA fragments ofapproximately 700 bp in length are inserted into a holding vector suchas pUC19 at the BamHI and EcoRI sites to give the pL-vFcγ4 plasmid. Thesequence of the gene is confirmed by DNA sequencing.

To prepare the hG-CSF-L-vFcy_(γ4) fusion gene, the hG-CSF fragment isexcised from the phGCSF plasmid with HindIII and BamHI and then insertedto the 5′-end of the peptide linker in the pL-vFcγ4 plasmid to give thephG-CSF-L-vFcγ4 plasmid. This fusion gene comprising hG-CSF, a 16-aminoacid Gly-Ser peptide linker and the Fc_(γ4) variant gene is theninserted at the HindIII and EcoRI sites of a mammalian expressionvector, such as pcDNA3 (Invitrogen), as described for thehG-CSF-L-vFc_(γ2) fusion protein. The final expression vector plasmid isdesignated as phGFP4. FIG. 2B shows a fusion gene containing sequencesencoding hG-CSF, a 16-amino acid peptide linker(GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer), and the Fc_(γ4)variant with Ser228Pro and Leu235Ala mutations, and its correspondingamino acid sequence (SEQ ID NO: 20).

3. Construction of the Gene Encoding the hG-CSF-L-vFc_(γ1)Fusion Protein

The hinge domain of human IgG1 heavy chain contains 15 amino acidresidues (GluProLysSerCysAspLysThrHisThrCysProProCysPro, SEQ ID NO: 24)including 3 cysteine residues. Out of these 3 cysteine residues, the2^(nd) and 3^(rd) are involved in the formation of disulfide bondingbetween two heavy chains. The 1^(st) cysteine residue is involved in thedisulfide bonding to the light chain of IgG. Since there is no lightchain present in the Fc fusion protein molecule, this cysteine residuemay pair with other cysteine residues, leading to nonspecific disulfidebonding. The hinge domain of Fc_(γ1) can be truncated to eliminate the1^(st) cysteine residue (AspLysThrHisThrCysProProCysPro). The geneencoding the Fc_(γ1) region is obtained by reverse transcription and PCRusing RNA prepared from human leukocytes and appropriate 5′ primer (SEQID NO:13) and 3′ primer (SEQ ID NO:4). Resulting DNA fragmentscontaining the truncated hinge and complete sequences of CH2 and CH3domains of Fc_(γ1) is used as the template to generate the Fc_(γ1)variant with Leu234Val, Leu235Ala, and Pro331 Ser mutations (vFc_(γ1)),and its corresponding amino acid sequence (SEQ ID NO: 22).

One way to incorporate these mutations is as follows: two segments areproduced and then assembled by using the natural Fc_(γ1) as the templatein overlapping PCR. The 5′ segment is generated by using SEQ ID NO:14 asthe 5′ primer and SEQ ID NO:5 as the 3′ primer. This 5′ primer containsthe Leu234Val, Leu235Ala mutations and the 3′ primer contains thePro331Ser mutation. The 3′ segment is generated by using SEQ ID NO:6 asthe 5′ primer and SEQ ID NO:4 as the 3′ primer. These 5′ and 3′ segmentsare then joined at the region covering the Pro331Ser mutation by usingSEQ ID NO:14 as the 5′ primer and SEQ ID NO:4 as the 3′ primer. Thisamplified fragment of approximately 650 bp in length, together with asynthetic oligonucleotide of 55 bases (SEQ ID NO:15) containingLeu234Val and Leu235Ala, are joined in PCR by using SEQ ID NO:16 as the5′ primer and SEQ ID NO:4 as the 3′ primer. The SEQ ID NO:16 primercontains sequences encoding a 16-amino acid Gly-Ser peptide linkerincluding the BamHI site. The resulting DNA fragments of approximately700 bp in length are inserted into a holding vector such as pUC19 at theBamHI and EcoRI sites to give the pL-vFcγ1 plasmid. The sequence of thegene is confirmed by DNA sequencing.

To prepare the hG-CSF-L-vFc_(γ)1 fusion gene, the hG-CSF fragment isexcised from the phGCSF plasmid with HindIII and BamHI and inserted tothe 5′-end of the peptide linker in the pL-vFcγ1 plasmid to give thephG-CSF-L-vFcγ1 plasmid. The fusion gene comprising hG-CSF, a 16-aminoacid Gly-Ser peptide linker, and the Fc_(γ1) variant gene is theninserted at the HindIII and EcoRi sites of a mammalian expressionvector, such as pcDNA3 (Invitrogen), as described for thehG-CSF-L-vFc_(γ2) fusion protein. The final expression vector plasmid isdesignated as phGFP1. FIG. 2C shows a fusion gene containing sequencesencoding hG-CSF, a 16-amino acid peptide linker(GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer), and the Fc_(γ1)variant with Leu234Val, Leu235Ala and Pro331Ser mutations), and itscorresponding amino acid sequence (SEQ ID NO: 22).

3. Expression of the Fusion Protein in Transfected Cell Lines

Two different rhG-CSF have been produced: a glycosylated form producedin Chinese Hamster Ovary (CHO) cells and a nonglycosylated form producedin bacterial cells. Glycosylated rhG-CSF contains O-linkedoligosaccharides attached to the threonine amino acid residue atposition 133, accounting for approximately 4% of its molecular weight.The carbohydrate chain contributes to the stabilization of the proteinmolecule by suppressing polymerization and conformational changes(Oh-eda et al., J. Biol. Chem., 265:11432–11435, 1990). In in vitrostudies using rhG-CSF, the glycosylated form produced in CHO cells isbiologically more active than the nonglycosylated form produced inbacteirial cells (Nissen, Eur. J. Cancer, 30A Suppl 3:S12–S14, 1994).Furthermore, rhG-CSF derived from CHO cells was shown to beindistinguishable from its natural counterpart in terms of structuralcharacteristics and biological activity (Kubota et al., Biochem.(Tokyo),107:486–492, 1990). In randomized crossover studies in healthyvolunteers, glycosylated rhG-CSF has been found to be 25 to 30% morepotent than the nonglycosylated rhG-CSF on a weight for weight basis inthe mobilization of peripheral blood progenitor cells (see, for example,Hoglund, Med. Oncol., 15:229–233, 1998; Hoglund et al., Eur. J.Haematol., 59:177–183, 1997). To obtain the protein most suitable forclinical use, the hG-CSF-L-vFc fusion protein will be produced in CHOcells as follows.

The recombinant phGFP1, phGFP2 or phGFP4 expression vector plasmid istransfected into a mammalian host cell line to achieve the expression ofthe hG-CSF-L-vFc fusion protein. For stable high levels of expression, apreferred host cell line is CHO cells deficient in the DHFR enzyme (see,for example, U.S. Pat. No. 4,818,679). A preferred method oftransfection is electroporation. Other methods, including calciumphosphate co-precipitation, lipofectin, and protoplast fusion, can alsobe used. For electroporation, 10 μg of plasmid DNA linearized with BspCIis added to 2 to 5×10⁷ cells in a cuvette using Gene PulserElectroporator (Bio-Rad Laboratories, Hercules, Calif.) set at anelectric field of 250 V and a capacitance of 960 μFd. Two days followingthe transfection, the media are replaced with growth media containing0.8 mg/ml of G418. Transfectants resistant to the selection drug aretested for the secretion of the fusion protein by anti-human IgG FcELISA. Quantitation of the expressed fusion protein can also be carriedout by ELISA using anti-hG-CSF assays. The wells producing high levelsof the Fc fusion protein are subcloned by limiting dilutions on 96-welltissue culture plates.

To achieve higher levels of the fusion protein expression,co-amplification is preferred by utilizing the gene of DHFR that can beinhibited by the MTX drug. In growth media containing increasingconcentrations of MTX, the transfected fusion protein gene isco-amplified with the DHFR gene. Transfectants capable of growing inmedia with up to 1 μg/ml of MTX are again subcloned by limitingdilutions. The subcloned cell lines are further analyzed by measuringthe secretion rates. Several cell lines yielding secretion rate levelsover about 10, preferably about 30 μg/10⁶ [i.e. million]cells/24 h, areadapted to suspension culture using serum-free growth media. Theconditioned media are then used for the purification of the fusionprotein.

5. Purification and Characterization of the Fusion Protein

Conditioned media containing the fusion protein are titrated with 1 NNaOH to a pH of 7 to 8 and filtered through a 0.45 micron cellulosenitrate filter. The filtrate is loaded onto a Prosep A columnequilibrated in phospate-buffered saline (PBS). After binding of thefusion protein to Prosep A, the flow-through fractions are discarded.The column is washed with PBS until OD at 280 nm is below 0.01. Thebound fusion protein is then eluted with 0.1 M citrate buffer at pH3.75. After neutralizing with 0.4 volume of 1 M K₂HPO₄, fractionscontaining purified protein are pooled and dialyzed against PBS. Thesolution is then filtered through a 0.22 micron cellulose nitrate filterand stored at 4° C. The molecular weight of purified hG-CSF-L-vFcprotein is in the range of 90 and 110 kDa by SDS-PAGE under non-reducingconditions. Under reducing conditions, the purified protein migratesaround approximately 50 kDa. The fusion protein is quantitated by BCAprotein assay using BSA as the standard.

6. In Vitro Biological Assays

Supernatants of transfectants or purified proteins can be tested fortheir ability to stimulate the proliferation of murine myeloblasticNFS-60 cells (Shirafuji et al., Exp. Hematol., 17:116–119, 1989). NFS-60cells are responsive to rhG-CSF but not to rhGM-CSF or hM-CSF. The cellsare maintained in growth medium (RPMI-1640 medium containing 10% FCS andmurine IL-3 at 1 ng/ml). Log phase NFS-60 cells are collected and washedwith assay medium (growth medium without murine IL-3). A total of 1×10⁴cells per sample of NFS-60 in 50 μl is added to each well of a 96-welltissue culture plate. The cells are incubated with 50 μl of assay mediacontaining various concentrations of the hG-CSF-L-vFc fusion protein orthe rhG-CSF control from 0.01 to 100 nM each. The plate is kept at 37°C. and 5% CO₂ in a humidified incubator for 4 days before 10 μl of MTT(2.5 mg/ml in PBS) is added to each well. After 4 h, the cells andformazan are solubilized by adding 100 μl per well of 10% SDS in 0.01 NHCl. The plate is then read at 550 nm with the reference beam set at 690nm. The OD reading is plotted against the concentration of rhG-CSF orthe fusion protein. The inflection point of the sigmoidal curverepresents the concentration at which 50% of the maximal effect, ED50,is induced. The biological activity of hG-CSF-L-vFc relative to that ofrhG-CSF can therefore be compared quantitatively. Preferably, therecombinant fusion proteins should be characterized by and exhibit anenhanced activity of at least 2 fold (2×) relative to that of rhG-CSF ona molar basis. In one embodiment of the present invention, the specificactivity of the hG-CSF-L-vFc fusion protein is in the range of about 1.5to about 6.0×10⁹ units/μmole, compared to about 0.75 to about 3.0×10⁹units/μmole for rhG-CSF based on this cell proliferation assay.

Supernatants of transfectants or purified proteins can also be testedfor their ability to stimulate the proliferation and differentiation ofhuman bone marrow progenitor cells to form colonies,granulocyte-macrophage colony forming unit (CFU-GM). The procedure is asfollows. Light-density cells from human bone marrow centrifuged overFicoll-Pague are washed and resuspended at 1×10⁶ cells/ml in Iscove'smodified Dulbecco's medium (IMDM) containing 5% FCS. These cellscontaining enriched progenitor cells are incubated in a tissue culturedish overnight at 37° C. and 5% CO₂ to remove all adherent cellsincluding monocytes, macrophages, endothelial cell, and fibroblasts.Cells in suspension are then adjusted to 1×10⁵ cells/ml in IMDMcontaining 5% FCS. For the assay, 0.3 ml of cells, 15 μl of stem cellfactor at 20 μg/ml, 2.4 ml of methylcellulose, and 0.3 ml of mediacontaining several concentrations of hG-CSF-L-vFc (or rhG-CSF control)are mixed. One ml of this cell mixture is plated on a 35-mm petri dish.The dishes are then kept at 37° C. and 5% CO₂ for 10 to 14 d before thecolonies are counted. A dose responsive curve can be plotted against theconcentrations of hG-CSF-L-vFc relative to those of rhG-CSF.

7. In Vivo Pharmacokinetic Studies in Rats

Fisher rats (Harlan Bioproducts for Science, Indianapolis, Ind.) with anaverage body weight of about 500 g are injected i.v. through the tailvein or s.c. with 100 units of rhG-CSF or the hG-CSF-L-vFc fusionprotein. An equal volume of PBS is injected as a control. Serial 0.5-mlsamples are taken through retro-orbital bleeds at different points (0,0.2, 1, 4, 24, 48, 96, and 168 h) after injection. There are 3 rats foreach time point. Whole blood is collected into tubes containinganticoagulant, cells are removed, and plasma is frozen at −70° C. untilassay is carried out.

Serum samples are used for NFS-60 cell assays, which measure theactivity of hG-CSF-mediated cell proliferation. A total of 1×10⁴ cellsper sample of NFS-60 in 50 μl is added to each well of a 96-well tissueculture plate. The cells are incubated with 50 μl of assay mediacontaining various concentrations of titrated blood samples. The plateis kept at 37° C. and 5% CO₂ in a humidified incubator for 4 days.Viable cells are stained with 10 μl of MTT (2.5 mg/ml in PBS). After 4h, the cells and formazan are solubilized by adding 100 μl per well of10% SDS in 0.01 N HCI. The plate is then read at 550 nm with thereference beam set at 690 nm. The activities of serum samples areplotted against time points for the calculation of the circulation time.The activity of hG-CSF-L-vFc decreases much slower than that of therhG-CSF control, indicating the longer circulating half-life of thefusion protein in rats.

The examples described above are for illustration purposes only. Theyare not intended and should not be interpreted to limit either the scopeor the spirit of this invention. It can be appreciated by those skilledin the art that many other variations or substitutes can be used asequivalents for the purposes of this invention, which is defined solelyby the written description and the following claims.

TABLE 1 Sequences of Oligonucleotides. SEQ ID NO:15′-cccaagcttcccagacccatggctggacct-3′ SEQ ID NO:25′-cggatccgggctgggcaaggtggcgta-3′ SEQ ID NO:3 5′-gagcgcaaatgttgtgtcga-3′SEQ ID NO:4 5′-ggaattctcatttacccggagacaggga-3′ SEQ ID NO:55′-tggttttctcgatggaggctgggaggcct-3′ SEQ ID NO:65′-aggcctcccagcctccatcgagaaaacca-3′ SEQ ID NO:75′-cggatccggtggcggttccggtggaggcggaagcggcggtggaggatcagagcgcaaatgttgtgtcga-3′SEQ ID NO:8 5′-gagtccaaatatggtccccca-3′ SEQ ID NO:95′-ggaattctcatttacccagagacaggga-3′ SEQ ID NO:105′-cctgagttcgcggggggacca-3′ SEQ ID NO:115′-gagtccaaatatggtcccccatgcccaccatgcccagcacctgagttcgcggggggacca-3′ SEQID NO:125′-cggatccggtggcggttccggtggaggcggaagcggcggtggaggatcagagtccaaatatggtccccca-3′SEQ ID NO:13 5′-gacaaaactcacacatgccca-3′ SEQ ID NO:145′-acctgaagtcgcggggggaccgt-3′ SEQ ID NO:155′-gacaaaactcacacatgcccaccgtgcccagcacctgaagtcggggggaccgt-3′ SEQ ID NO:165′-cggatccggtggcggttccggtggaggcggaagcggcggtggaggatcagacaaaactcacacatgccca-3′

1. A recombinant hG-CSF-L-vFc fusion protein comprising hG-CSF, apeptide linker, and a human IgG Fc variant, wherein the human IgG Fcvariant comprises a hinge, CH2, and CH3 domains of human IgG1 withLeu234Val, Leu235Ala, and Pro331Ser mutations as SEQ ID NO
 22. 2. Therecombinant hG-CSF-L-vFc fusion protein of claim 1, wherein the peptidelinker (i) comprises about 20 or fewer amino acids; (ii) is presentbetween hG-CSF and the human IgG Fc variant; and (iii) comprises two ormore amino acids selected from the group consisting of glycine, serine,alanine, and threonine.
 3. The recombinant hG-CSF-L-vFc fusion proteinof claim 1, wherein the hG-CSF-L-vFc fusion protein is characterized byan enhanced in vitro biological activity of at least 2 fold relative tothat of rhG-CSF on a molar basis.
 4. A CHO-derived cell line producingthe hG-CSF-L-vFc fusion protein of claim 1 in the cell line's growthmedium in excess of 10 μg per million cells in a 24 hour period.
 5. TheCHO-derived cell line producing the hG-CSF-L-vFc fusion protein of claim4 in the cell line's growth medium in excess of 30 μg per million cellsin a 24 hour period.
 6. A method for making a recombinant fusion proteincomprising hG-CSF, a flexible peptide linker, and a human IgG Fcvariant, which method comprises: (a) generating a CHO-derived cell lineby transforming the CHO cell line with a gene encoding the recombinantfusion protein comprising hG-CSF; (b) growing the cell line underconditions sufficient for expressing the recombinant fusion protein inthe cell line's growth medium at a rate of in excess of 10 μg permillion cells in a 24 hour period; and (c) purifying the expressedprotein from step (b), wherein the recombinant fusion protein ischaracterized by an enhanced in vitro biological activity of at least 2fold relative to that of rhG-CSF on a molar basis; and wherein the humanIgG Fc variant comprises a hinge, CH2, and CH3 domains of human IgG1with Leu234Val, Leu235Ala, and Pro331Ser mutations as SEQ ID NO
 22. 7.The method of claim 6, wherein in step (b) growing the cell line underconditions sufficient for expressing the recombinant fusion protein inthe cell line's growth medium at a rate of in excess of 30 μg permillion cells in a 24 hour period.
 8. The method of claim 6, wherein theflexible peptide linker (i) comprises about 20 or fewer amino acids;(ii) is present between hG-CSF and the human IgG Fc variant; and (iii)comprises two or more amino acids selected from the group consisting ofglycine, serine, alanine, and threonine.
 9. The method of claim 8,wherein in step (b) growing the cell line under conditions sufficientfor expressing the recombinant fusion protein in the cell line's growthmedium at a rate of in excess of 30 μg per million cells in a 24 hourperiod.
 10. A method for making a recombinant fusion protein comprisinghG-CSF, a flexible peptide linker, and a human IgG Fc variant, whichmethod comprises: (a) generating a CHO-derived cell line by transformingthe CHO cell line with a gene encoding the recombinant fusion proteincomprising hG-CSF; (b) growing the cell line under conditions sufficientfor expressing the recombinant protein in the cell line's growth mediumat rate of in excess of 10 μg per million cells in a 24 hour period; and(c) purifying the expressed protein from step (b), wherein therecombinant fusion protein is characterized by an enhanced in vitrobiological activity of at least 2 fold relative to that of rhG-CSF on amolar basis; wherein the flexible peptide linker (i) comprises about 20or fewer amino acids; (ii) is present between hG-CSF and the human IgGFc variant; and (iii) comprises two or more amino acids selected fromthe group consisting of glycine, serine, alanine, and threonine; andwherein the human IgG Fc variant comprises a hinge, CH2, and CH3 domainsselected from the group consisting of human IgG1 with Leu234Val,Leu235Ala, and Pro331Ser mutations as SEQ ID NO
 22. 11. The method ofclaim 10, wherein in step (b) growing the cell line under conditionssufficient for expressing the recombinant fusion protein in the cellline's growth medium at a rate of in excess of 30 μg per million cellsin a 24 hour period.